Accurate sizing of red blood cells is an important measurement in the determination of various pathologic hematologic conditions. Normal red cell diameter is typically 7.5-8.3.mu.. Iron deficiency anemias and thalassemias result in microcytosis, where red cell diameters may be as small as 6.mu.. Megaloblastic anemias, pernicious anemia, and folate and vitamin B.sub.12 deficiencies, on the other hand, lead to macrocytosis, where red cell diameters can be 9.mu. or larger. Price-Jones, in 1933, was the first to quantify, by direct microscopic measurement, variations in red blood cell sizes. The Price-Jones histogram of frequency of occurrence versus cell size has been used to relate these variations to differences in the physiology and pathology of stress and disease situations. Because these direct measurements are arduous and involve relatively small numbers of cells, this procedure has largely been ignored as a routine quantitative parameter by hematology laboratories. With the advent of electronic cell counters and analyzers, the mean corpuscular volume (MCV) has become the standard index for measuring the average size (volume) or thousands of cells in a matter of seconds. The problem with MCV, however, is that it only provides an average value and gives little information concerning the actual size and/or shape of individual cells.
It is, accordingly, a primary object of the present invention to provide faster, more accurate, and more reliable methods of investigating red blood cell abnormalities.
It is a related object to utilize the principles of optical flow cytometry to create a red cell abnormality screening vehicle.
Several investigators have reported using slit-width or time-of-flight ("TOF") measurements of impedance, light scatter, or fluorescence pulses generated by electronic cell counters to estimate cell size. These measurements are fairly accurate for spherical particles, but less advantageous for real cells which are of varying shapes and refractive indices. Leary, Todd, Wood and Jett, in 1979, increased the resolution over TOF analyses by looking at pulse risetime measurements, which they found to also be highly linear functions of diameter for microspheres. As a result of this work, James F. Leary and Paul Todd have a U.S. Pat. No. 4,263,508, issued on Apr. 21, 1981, entitled Pulse Edge Measurement for Determining Particle Dimensional Characteristics.
It is another object of the present invention to utilize the dynamics of cell counters, but more efficiently to rely on the wealth of information thereby made available. For example, it has been found that in the normal processing of samples in a commercially available clinical optical flow cytometry instrument (such as the one available from Ortho Diagnostic Systems Inc. of Raritan, NJ and Westwood, MA, the assignee hereof, under the trade name ELT-8) as many as thirty-three relevant parameters may be indexed. Clearly, it is desirable to base red cell abnormality investigations on an optimum selection of these parameters, considering the need for accuracy offset against the time and cost of complex processing.